水牛黑色素皮质素1受体基因克隆、生物信息学分析及表达模式研究

Cloning,Bioinformatics and Expression Pattern Analysis of Buffalo MC1R Gene

本试验旨在对水牛黑色素皮质素1受体(MC1R)基因进行克隆、生物信息学分析及表达模式研究。参考牛MC1R基因(GenBank登录号:JN123363.1)序列设计引物,以本地沼泽水牛、白沼泽水牛、摩拉水牛和黄牛基因组DNA为模板,应用PCR方法扩增克隆MC1R基因片段并进行测序分析。运用QRT-PCR方法检测摩拉水牛、沼泽水牛、白沼泽水牛和黄牛皮肤组织中MC1R基因的表达模式,并通过Western blotting方法检测沼泽水牛和白沼泽水牛MC1R基因的蛋白表达差异。结果表明,应用PCR方法成功克隆了水牛MC1R基因,其编码区全长954bp,共编码317个氨基酸。测序分析后发现沼泽水牛、白沼泽水牛、摩拉水牛和黄牛MC1R基因的核苷酸序列和氨基酸序列相似性很高。沼泽水牛与白沼泽水牛在476、618、881、930和931bp位点上分别发生T→C、G→C、G→A、G→A和A→G突变,导致了沼泽水牛和白沼泽水牛第159位氨基酸由丝氨酸变成苯丙氨酸,第310位氨基酸由谷氨酸变成丙氨酸,第294位氨基酸由天冬氨酸变成丙氨酸,发生了非同义突变。QRT-PCR结果发现,MC1R基因在摩拉水牛、沼泽水牛和黄牛皮肤组织中的相对表达量均显著高于白沼泽水牛(P<0.05);Western blotting分析结果显示,沼泽水牛皮肤组织中MC1R蛋白的表达量高于白沼泽水牛。综上所述,白沼泽水牛MC1R基因的编码区发生氨基酸位点突变,且相对表达量和蛋白表达量均低于沼泽水牛,推测此为白沼泽水牛体内合成的黑色素缺失而导致毛色白化的主因。

The aim of this study was to clone and analyze the expression pattern of buffalo MC1 R gene.A pair of specific primers was designed according bovine MC1Rsequence（GenBank accession No.：JN123363.1）,with genome DNA of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle as template,MC1 Rwas amplified by PCR.Then relative expression level of MC1 Rgene of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle was analyzed using QRT-PCR,and protein expression was detected by Western blotting method.The results showed that the 954 bp coding region of buffalo MC1 Rgene was successfully cloned and sequenced,which code for 317 amino acids.The MC1 Rgene nucleotide sequences and amino acid sequences of swamp buffalo,White swamp buffalo,Murrah buffalo and Yellow cattle were highly conserved.Five polymorphic sites were found between White swamp buffalo and swamp buffalo ofMC1 Rgene,including 476T→C,618G→C,881G→A,930G→A and 931A→G,which caused three nonsynonymous mutation sites of Phe159 Ser,Glu310Ala and Asp294 Ala.The QRT-PCR result showed that relative expressions of MC1 Rgene of Murrah buffalo,swamp buffalo and Yellow cattle were significant higher than that of White swamp buffalo（P〈0.05）.The Western blotting results revealed that MC1 Rprotein expression level in swamp buffalo was higher than that of White swamp buffalo.In conclusion,there were amino acids mutation in White swamp buffalo MC1 Rgene,and MC1 Rgene relative expression of White swamp buffalo was lower than that of other buffalo,which was the main reason of lacking of melanin production in White swamp buffalo.